Non-small cell lung cancer (NSCLC), although considered non-immunogenic, is often resistant to chemotherapy agents during the course of treatment in clinical patients. Melittin (C₁₃₁H₂₂₉N₃₉O₃₁, CAS: 20449-79-0), the major component of honey bee venom, is a promising anticancer drug. However, the mechanism employed by melittin to reverse chemotherapy resistance of NSCLC cells remains unknown. In this study, the Cell Counting Kit 8, ethynyl deoxyuridine assay, and other assays were utilized to elucidate the melittin effects upon cell proliferation. Proteomics, lung cancer (LC) tissue chip, and Western blot analysis were used to identify potential targets of melittin. A549/DDP cells were employed to investigate the melittin effects against cisplatin resistance. Also, an in vivo animal experiment was conducted to further clarify the regulatory function of melittin towards cisplatin resistance of A549/DDP cells. Results showed that melittin inhibited malignant progression of A549/DDP cells by down-regulation of pyruvate dehydrogenase kinase 3 (PDK3)-mediated aerobic glycolysis and inhibition of heat shock factor 1 (HSF1) expression. The therapeutic effect of melittin was increased by combination with KNK437 and impaired chemotherapy resistance regarding A549/DDP cells via reversing aerobic glycolysis. The in vivo experiments confirmed that melittin incremented A549/DDP cell cisplatin sensitivities. Collectively, the data suggested that melittin suppressed aerobic glycolysis by regulating HSF1/PDK3, which incremented cisplatin sensitivity of A549/DDP cells. It may provide a new treatment method for chemotherapy resistance in clinical NSCLC patients.